Fluorescence detection of free radicals by nitroxide scavenging.

The fluorescence quantum yield of 4-(1-napthoyloxy)-2,2,6,6-tetramethylpiperidine-1-oxyl (I) in acetonitrile and hexane is 55 and 30-fold lower, respectively, than those of diamagnetic analogs. Experiments described herein demonstrate that this property makes possible the fluorescence detection of radical scavenging reactions in which the paramagnetic nitroxide-substituted naphthalene is converted to a diamagnetic N-alkoxy derivative. 2-Cyanopropyl free radicals were generated by the thermal decomposition of azobisisobutyronitrile (AIBN) in cyclohexane or in acetonitrile containing I. The fluorescence intensity of the sample increased proportionally to the decrease in its ESR signal intensity, indicating the conversion of the paramagnetic nitroxide to the diamagnetic product. The linear relationship between the increase in fluorescence intensity and decrease in ESR signal intensity shows that the changes in the fluorescence intensity can serve as a sensitive means for optically detecting radicals.

MNDO calculations of highly mutagenic (chloromethyl)benzo[a]pyrenes.

(Chloromethyl)benzo[a]pyrenes (ClMeB[a]P) are among the most potent direct-acting mutagens (i.e., not requiring metabolic activation) detected by the Ames assay using strain TA98 and TA100. We wanted to determine if molecular quantum mechanics would yield insights into the reactivity and mutagenicity of these compounds. Calculations using the MNDO semiempirical method (MOPAC program) were carried out on a series of ClMeB[a]P. The experimental rates of solvolysis of ClMeB[a]P in 50% aqueous acetone correlated well (r = -0.95) with the differences between the calculated heats of formation of ClMeB[a]P and those of their respective carbocations, supporting the hypothesis that ClMeB[a]P react via carbocations. None of the calculated parameters correlated with mutagenicity. Thus, the prediction of mutagenicity of ClMeB[a]P in the Ames assay is more complex than the simple formation of carbocation intermediates.

Mutagenicity and induction of sister chromatid exchange by optically active enantiomers of secondary butyl methanesulfonate.

This report describes experiments in which a chiral alkyl methanesulfonate was used to investigate possible mechanisms by which alkylating agents cause their mutagenic, cytotoxic, and clastogenic effects. Optically active enantiomers and the racemic mixture of 2-butyl methanesulfonate (2-BMS) were cytotoxic and mutagenic in Chinese hamster V79 cells and in AS52 cells and mutagenic in Salmonella typhimurium strains TA100 and TA1535 (without the addition of exogenous metabolizing systems). Within the experimental uncertainties, the cytotoxicity and mutagenicity curves were the same for the R and S enantiomers and for the racemic mixture. The 2-BMS isomers were cytotoxic and induced sister chromatid exchanges (SCE) in CHO-K1-BH4 cells. The cytotoxicity curve was similar to that observed with V79 and AS52 cells. The induction of SCE was linear between 1 and 6 mM 2-BMS with no differences discernable between the isomers. The results can be interpreted two ways. The first interpretation is that 2-BMS reacts via a carbocation, and the second interpretation involves an SN2 reaction of 2-BMS with DNA. The latter interpretation suggests that the mechanisms of mutagenesis, cytotoxicity, or the induction of SCE cannot distinguish between small (four-carbon) optically active DNA adducts. We favor the second interpretation because of solvolysis experiments showing the complete inversion of configuration of optically active 2-octyl methanesulfonate (2-OMS, Weiner and Sneen: J American Chemical Society 87:287-291, 1965). While we assume that optically active 2-BMS will react using the same mechanism as chiral 2-OMS, we cannot exclude the possibility that 2-BMS reacts via a carbocation intermediate.